We are investigating the molecular mechanisms which control the individual and total concentrations of hemoglobins in human erythrocytes. The study of the control of hemoglobin levels has direct relevance to various hemoglobinopathies, especially thalassemia and sickle cell disease. For our experimental system, we are using the K562 human leukemic cell line, as well as peripheral blood from individuals with sickle cell disease. We are studying the effects of short-term and long-term exposure of these cells to 5-azacytidine and hemin or their phenotype and the factors that control globin gene transcription. Adult beta-mRNA expression remains undetectable, yet we have found a constitutive level of another adult type hemoglobin, delta-mRNA, whose expression is inducible both with hemin and 5-azacytidine. Because of the close sequence homologies between the delta- and beta-globin genes, experiments are underway to examine whether changes in the delta-promoter sequence may alter important protein binding sites and thereby result in the low levels of delta-globin gene expression. A 500 bp region 5' to the delta globin CAP site has been cloned into a chloramphenicol acetyl transferase (CAT) reporter plasmid. Preliminary studies demonstrate that this reporter system when transfected into K562 give quantitatively similar results (when compared to beta-CAT and epsilon- CAT) as were obtained using S-1 nuclease mapping. Identification of these putative protein(s) binding sites may not only provide important information on the regulation of the minor hemoglobin synthesis but may allow for the characterization of trans-acting factor(s) responsible for beta-globin gene expression. Development of this functional assay system utilizing the various CAT constructions may facilitate such identification. Investigations are also currently underway to determine the effects of copper and thyroid hormone, which have been recognized clinically to modulate HbA2 levels in patients.